The present invention relates to the field of immunology and is particularly concerned with a novel lactoferrin receptor protein isolated and purified from bacterial pathogens and methods of production, and uses thereof.
Most living organisms require a continuous supply of iron to maintain growth and thus have evolved efficient mechanisms for acquisition of iron under conditions of limitation (ref. 1xe2x80x94Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). A common mechanism found in many bacterial species involves the synthesis and secretion of small iron-chelating molecules, siderophores, which complex with iron and are subsequently bound and internalized via specific receptors at the bacterial surface (ref. 2). This mechanism is effective in a wide variety of environments and is often found in bacterial species that are present in a variety of ecological niches.
The vertebrate host provides an iron-restricted environment to potential bacterial pathogens, largely due to the sequestration of extracellular iron by the host iron-binding glycoproteins, namely transferrin (Tf) and lactoferrin (Lf). Although a siderophore-mediated mechanism should be effective in this setting, some bacterial species have evolved alternative mechanisms of iron acquisition that are adapted to their particular host. Thus, some members of the Pasteurellaceae and Neisseriaceae are capable of specifically binding and effectively acquiring iron from the host Tf and in some cases, Lf. This is mediated by receptors present at the bacterial surface whose expression is induced by restricting the level of available iron in the medium.
Receptors that are specific for Tf from the host (ref. 3) have been identified in a variety of important human and veterinary pathogens as well as some commensal species (Table 1). To date, Tf receptors have only been identified in bacterial species within the Pasteurellaceae and Neisseriaceae. In most species the Tf receptor has been shown to consist of two proteins, Tf-binding protein I (Tbp1) and Tf-binding protein 2 (Tbp2). The genes encoding these proteins have been cloned from Neisseria meningitidis (ref. 13), N. gonorrhoeae (refs. 14 and 15), Haemophilus influenzae (ref. 16) and Actinobacillus pleuropneumoniae (refs. 17 and 18). The predicted amino acid sequences of Tbp2 proteins reveal homology to the lipoprotein signal peptidase recognition site suggesting that it is lipid-modified and retains an association with the outer membrane via its lipid tail. Lipidation of Tbp2 has been confirmed by labelling (refs. 13 and 19) and evidence is accumulating that Tbp2 is largely surface exposed (refs. 20 and 21). Isogenic mutants deficient in the production of Tbp2 demonstrate severely limited ability to utilize transferrin as a sole iron source during in vitro growth studies, suggesting Tbp2 plays an important, albeit not essential role in iron acquisition (refs. 14, 16, 22).
Based on its homology with other TonB-dependant outer membrane proteins Tbp1 receptor proteins are believed to have several regions which span the outer membrane (ref. 23) (see ref. 35 for a topological model that can be applied to Tbp1). Similarly, based on the results obtained with the FepA receptor (ref. 25), Tbp1 is thought to act as a gated pore which allows the passage of iron from the transferrin and lactoferrin molecules, which are themselves not internalized, to the periplasm where it is bound by the ferric iron-binding protein, FbpA. Two additional proteins FbpB and FbpC are believed to be involved in the transport of iron across the cytoplasmic membrane.
The transport of iron across the outer membrane is believed to occur in a TonB-dependent manner, in that amino acid sequences referred as the xe2x80x9cTonB boxxe2x80x9d, located in a number of TonB-dependent outer membrane receptor proteins have also been identified in Tbp1. The inability to utilize human transferrin following insertional inactivation of the H. influenzae TonB homologue clearly supports this theory. In addition, mutants in which the Tbp1 protein has been insertionally inactivated are unable to utilize transferrin as a sole iron source, supporting its essential role in iron acquisition from transferrin.
Bacterial lactoferrin receptors have only been described for human pathogens in the Neisseriaceae, and were thought to consist of a single protein, Lbp1. Amino acid sequence analysis of the Lbp1 protein shows high homology to Tbp1 (refs. 26, 35, 36), and isogenic mutant analysis deficient in Lbp1 suggests an essential role of Lbp1 in iron acquisition (refs. 24 and 28). Recent genetic evidence suggests that similar to the tbpBA operonic organization, an open reading frame is located immediately upstream of the lbpA structural gene which may encode a Tbp2 homologue, Lbp2 (ref. 35).
Properties of lactoferrin receptor proteins of bacterial pathogens indicate that these proteins have utility in diagnosis of and vaccination against diseases caused by such bacterial pathogens that produce lactoferrin receptor proteins or proteins capable of raising antibodies specifically reactive with lactoferrin receptor proteins.
It would be advantageous to provide purified lactoferrin receptor proteins (and methods of purification thereof) for use as antigens, immunogenic preparations, including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents.
The present invention is directed towards the provision of purified and isolated lactoferrin receptor protein from a bacterial pathogen having a molecular mass of about 70,000 and about 90,000 daltons wherein the molecular mass is determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
In accordance with one aspect of the invention, there is provided a lactoferrin receptor protein isolated and purified from a bacterial pathogen and having an apparent molecular mass of about 70,000 to about 90,000 daltons as determined by SDS-PAGE.
The bacterial pathogens from which the lactoferrin receptor protein may be isolated and purified include Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Moraxella bovis and Moraxella lacunata. The about 70 to about 90 kDa lactoferrin receptor protein is at least about 70 wt % pure, preferably at least about 90 wt % pure, and may be in the form of an aqueous solution thereof.
The lactoferrin receptor protein, sometimes referred to herein by the designation xe2x80x9cLbp2xe2x80x9d, may be provided in a form substantially free from a lactoferrin receptor protein having a molecular weight of about 100,000 to about 105,000 daltons, as determined by SDS-PAGE, sometimes referred to herein by the designation xe2x80x9cLbp1xe2x80x9d.
Alternatively, the novel lactoferrin receptor protein provided herein may comprise a mixture of the lactoferrin receptor proteins. In another aspect of the present invention, therefore, there is provided a composition comprising a first lactoferrin receptor protein (Lbp1) having a molecular weight of about 100,000 to about 105,000 and a second lactoferrin receptor protein having a molecular weight of between about 70,000 and about 90,000 daltons, wherein the molecular weights are determined by SDS-PAGE and the lactoferrin receptor proteins are isolated and purified from a bacterial pathogen, which may be any of the pathogens mentioned above.
Such composition may be at least about 70% pure by weight, preferably at least about 90% pure by weight. Such composition also may be provided substantially free from transferrin receptor proteins of the pathogen.
The present invention also provides an immunogenic composition comprising an immunoeffective amount of an active component, which may be the novel lactoferrin receptor protein, provided herein alone or together with Lbp1 along with a pharmaceutically acceptable carrier therefor. The immunogenic composition may be formulated as a vaccine for in vivo administration to a host to confer protection against diseases caused by a bacterial pathogen which produces a lactoferrin receptor protein having a molecular weight of about 70,000 to about 90,000 daltons, or that produces a protein capable of inducing antibodies in the host specifically reactive with the lactoferrin receptor protein. The immunogenic composition may be formulated as a microparticle capsule, ISCOM or liposome preparation. The immunogenic composition may be used in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces. Some targeting molecules include strain B12 and fragments of bacterial toxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.), and monoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barber et al). The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant. Suitable adjuvants for use in the present invention include, (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide and a lipoprotein. Advantageous combinations of adjuvants are described in copending U.S. patent application Ser. No. 08/261,194 filed Jun. 16, 1994, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference. The immunogenic composition may be substantially free from transferrin receptor protein of the pathogen.
The invention further includes an antibody specific for the novel lactoferrin receptor protein provided herein producible by immunizing a host with an immunogenic composition as provided herein.
In a further aspect of the invention, there is provided a method of generating an immune response in a host, comprising administering thereto an immuno-effective amount of the immunogenic composition as provided herein. The immune response may be a humoral or a cell-mediated immune response. Hosts in which protection against disease may be conferred include primates including humans.
The immune response which is generated may provide protection to the host against disease caused by a bacterial pathogen that produces a lactoferrin receptor protein having a molecular weight of between about 70,000 and about 90,000 daltons or that produces a protein capable of inducing antibodies in the host specifically reactive with the lactoferrin receptor protein.
The present invention provides, in an additional aspect thereof, a method for producing a vaccine comprising administering the immunogenic composition provided herein to a test host to determine an amount and a frequency of administration of the lactoferrin receptor protein to confer protection against disease caused by a bacterial pathogen that produces the lactoferrin receptor protein having a molecular weight between about 70,000 and about 90,000 or a protein capable of inducing antibodies in a host specifically reactive with the lactoferrin receptor protein; and formulating the lactoferrin receptor protein in a form suitable for administration to a treated host in accordance with said determined amount and frequency of administration. The treated host may be a human.
A further aspect of the invention provides a method of determining the presence in a sample, of antibodies specifically reactive with a lactoferrin receptor protein of a bacterial pathogen having a molecular weight of between about 70,000 and about 90,000 daltons, comprising the steps of:
(a) contacting the sample with the lactoferrin receptor protein as provided herein to produce complexes comprising the lactoferrin receptor protein and any said antibodies present in the sample specifically reactive therewith; and
(b) determining production of the complexes.
In a further aspect of the invention, there is provided a method of determining the presence, in a sample, of a lactoferrin receptor protein of a bacterial pathogen and having a molecular weight of between about 70,000 and about 90,000 daltons, comprising the steps of:
(a) immunizing a subject with the lactoferrin receptor protein as provided herein, to produce antibodies specific for the lactoferrin receptor protein;
(b) contacting the sample with the antibodies to produce complexes comprising any outer membrane protein present in the sample and said outer membrane protein specific antibodies; and
(c) determining production of the complexes.
A further aspect of the invention provides a diagnostic kit for determining the presence of antibodies in a sample specifically reactive with the lactoferrin receptor protein of a bacterial pathogen and having a molecular weight of between about 70,000 and about 90,000 daltons, comprising:
(a) the lactoferrin receptor protein as provided herein;
(b) means for contacting the lactoferrin receptor protein with the sample to produce complexes comprising the lactoferrin receptor protein and any said antibodies present in the sample; and
(c) means for determining production of the complexes.
The invention also provides a diagnostic kit for detecting the presence, in a sample, of a lactoferrin receptor protein of a bacterial pathogen and having a molecular weight of between about 70,000 and about 90,000, comprising:
(a) an antibody specific for the novel lactoferrin receptor protein as provided herein;
(b) means for contacting the antibody with the sample to produce a complex comprising the lactoferrin receptor protein and lactoferrin receptor protein-specific antibody; and
(c) means for determining production of the complex.
In an additional aspect, the present invention provides a method of producing a vaccine, comprising administering the immunogenic composition provided herein to a test host to determine an amount and a frequency of administration to confer protection against disease caused by a bacterial pathogen that produces a lactoferrin receptor protein having a molecular weight of between about 70,000 and about 90,000 daltons or a protein capable of inducing antibodies in a test host specifically reactive with the lactoferrin receptor protein; and formulating the immunogenic composition in a form suitable for administration to a treated host, which may be a human, in accordance with the determined amount and frequency of administration.
The present invention additionally provides a method of producing antibodies specific for a lactoferrin receptor protein having a molecular weight of between about 70,000 and about 90,000 daltons, comprising:
(a) administering the lactoferrin receptor protein provided herein to at least one mouse to produce at least one immunized mouse;
(b) removing B-lymphocytes from the at least one immunized mouse;
(c) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(d) cloning the hybridomas;
(e) selecting clones which produce anti-lactoferrin receptor protein antibody;
(f) culturing the anti-lactoferrin receptor protein antibody-producing clones; and then
(g) isolating anti-lactoferrin receptor protein antibodies from the cultures.
The present invention further includes procedures for isolating and purifying the novel lactoferrin receptor protein from a bacterial pathogen, either in a form free from Lbp1 or in a form of a purified mixture with Lbp1.
Accordingly, in another aspect of the invention, there is provided a method of producing a lactoferrin receptor protein from a bacterial pathogen, comprising the steps of:
(a) providing a solubilized membrane preparation from the bacterial pathogen containing lactoferrin receptor proteins, non-lactoferrin receptor proteins and other contaminants,
(b) complexing the lactoferrin receptor proteins with lactoferrin and purifying the resulting complexes substantially free from non-lactoferrin receptor proteins and the other contaminants, and
(c) separating from the complexes a lactoferrin receptor protein having a molecular weight of between about 70,000 and about 90,000 daltons.
This isolation and purification procedure may be carried out in any desired manner. The lactoferrin receptor proteins may further comprise a second lactoferrin receptor protein having a molecular weight of about 100,000 to about 105,000 daltons (i.e. lbp1).
In one embodiment, step (b) is effected by complexing the two lactoferrin receptor proteins with lactoferrin and separating the complexes from the membrane preparation, and step (c) produces a purified mixture of the two lactoferrin receptor proteins.
In another embodiment, the non-lactoferric receptor protein include transferrin receptor proteins of the pathogen, step (b) is effected by removing the second lactoferrin receptor protein (Lbp1) and the transferrin receptor proteins (Tbp1 and Tbp2) from the solubilized membrane preparation to provide a depleted membrane preparation, complexing the first-mentioned lactoferrin receptor protein with lactoferrin in the depleted membrane preparation and removing the complex so formed from the depleted membrane preparation.
In forming the complexes with the transferrin receptor protein(s), the transferrin may be coupled to a carrier molecule, which may be a Sepharose, such as cyanogen bromide activated CH-Sepharose. In one embodiment, the lactoferrin may be conjugated to biotin and the Sepharose in streptavidin-Sepharose to couple the lactoferrin to streptavidin.
In this application, the term xe2x80x9clactoferrin receptor proteinxe2x80x9d is used to define a family of lactoferrin receptor proteins of a variety of pathogens having, a molecular mass of between about 70,000 and 90 daltons and includes proteins having variations in their amino acid sequences including those naturally occurring in various strains of bacterial pathogens. In this application, a first protein or peptide is a xe2x80x9cfunctional analogxe2x80x9d of a second protein or peptide if the first protein is immunologically related to and/or has the same function as the second protein or peptide. The functional analog may be, for example, a fragment of the protein or a substitution, addition or deletion mutant thereof. The invention also extends to such functional analogs.
Advantages of the present invention include:
a method for isolating purified about 70,000 to about 90,000 daltons lactoferrin receptor protein of a bacterial pathogen that produces the lactoferrin receptor protein;
an isolated and purified lactoferrin receptor protein of a bacterial pathogen having a molecular weight of about 70,000 to about 90,000 daltons as determined by SDS-PAGE; and
diagnostic kits and immunological reagents for specific identification of bacterial pathogens and hosts infected thereby.